This invention relates generally to the identification of micro-organisms, and more particularly to a technique whereby a bacterium or virus may be identified by the characteristic band pattern of radioactive particles it generates under standardized conditions, the identified organism being quantified by the intensity of the pattern.
Identity is generally defined as "sameness in all that constitutes the objective reality of a thing". Though the concern of the invention is with the identification of micro-organisms, the problem of how best to carry out identification arises in those situations in which one must determine whether an unknown object, in all essential respects, matches a particular known object lying within a large collection of diverse known objects. In the context of microbiology, the term identification, as used herein, encompasses the determination of whether an unknown microbe falls within a particular class or genus of microbes as well as determining whether the microbe is of a particular type, species or strain.
The logical approach to the problem of identification is to find or create an identifier in the form of a symbol, signature, fingerprint or other indicia functioning to specify a particular object and no other in the collection. Thus while billions of individuals exist who differ from each other in sex, age, height, weight, shape, eye colour and in many other respects, a unique identifier is the individual's fingerprint. Hence if one has a collection or library of fingerprints covering all persons having a criminal record, by taking a fingerprint of an individual whose record is unknown, one can, by consulting the library, determine whether that fingerprint matches a particular print in the library.
Lacking an individual's fingerprint or identifier, identification would require matching of a large number of variables, such as sex, height, weight, etc., a relatively complicated procedure with uncertain results should only limited information be available as to the many variables involved. As will later be explained, it is the absence of an identifier for micro-organisms which presently complicates identification procedures in microbiology.
But the existence or creation of an identifier is only a partial solution to the problem of identification. An identifier is of limited practical value if it is not "machine readable" and therefore requires visual comparison to ascertain whether the identifier for an unknown unit matches a particular identifier in a library of known units.
Thus in the case of fingerprints, for many years the process of determining whether a match existed between a given fingerprint and one in a collection of known prints involved visual inspection carried out by fingerprint experts. However, use is currently made for this purpose of electro-optical scanners operating in conjunction with computers to automate fingerprint matching procedures.
Similarly, in the case of the large collection of diverse grocery and other items sold in supermarkets, in order to expedite the handling of these products at checkout counters and to facilitate inventory control and accounting procedures, use is now made of identifiers based on the Universal Product Code (UPC) whereby each product has printed thereon a UPC symbol. This symbol is in the form of a machine-readable bar code having digits identifying the manufacture of the product and digits identifying the product itself, so that each product has a unique bar code.
By electro-optically scanning the symbol on each purchased product and generating a corresponding signal, one is able in an associated computer whose memory has digitally stored therein the full range of supermarket products, to compare the signal derived from the scanned symbol with the stored information and to extract from the computer, when a match is found, the identity of the purchased product and its price.
Thus in the above-described situations, computer technology is exploited to facilitate identification procedures, use being made of machine-readable identifiers. But in the realm of microbiology in which identification plays a vital role, the identification procedures presently practised do not make use of identifiers for micro-organisms. Existing procedures require skilled laboratory personnel and are tedious and time-consuming; for when a specimen of an unknown micro-organism is developed, it must be visually examined under a microscope and then compared with available data regarding known forms.
These procedures represent a significant cost factor in microbiological studies, and by their very nature are subject to visual fatigue and human error. In recent years, the need for efficient and relatively rapid identification techniques has become more pressing because of the remarkable expansion of environmental and industrial microbiology, and genetic engineering. An interesting use of selected micro-organisms to degrade products of industrial organic syntheses is described in GB-PS 2,010,327A.
Techniques are available to determine the morphology of individual biological cells. Also well developed are procedures for growing or cultivating micro-organisms in the laboratory on nutrient material, some of these procedures requiring special conditions such as the absence of free oxygen. By incubating a nutrient agar-type medium, using the streak-plate or pour-plate method, cells are individually separated. In incubation, individual cells reproduce rapidly to generate a visible colony of cells, each colony being a pure sample of a single kind of micro-organism. In order to identify an unknown cell, existing techniques call for the use of high-magnification optical or electron microscopes. These procedures for the identification of bacteria are set forth in detail in chapter 5 of the text "Clinical Bacteriology"--Fifth Ed.--J. Stokes et al., published by Arnold (London) 1980.
Modern diagnostic medical practice is divided into specialized areas. Thus physicians who treat venereal disease must investigate and identify a class of micro-organisms that may differ from the class of concern to doctors specializing in blood or other disorders. To assist their doctors, hospitals make use of medical service laboratories which receive urine, blood and other specimens and are required to analyse and identify pathological organisms. Present service laboratory procedures for this purpose employ the traditional techniques of plating, staining and microscopy supplemented by a battery of individual tests for different types of micro-organism. These procedures entail a high order of technical skill and are labour-intensive. While most laboratory technicians are highly competent, the fact remains that mistaken identifications of pathological organisms are not uncommon in a period in which service laboratories operate under heavy work loads. And though in recent years automated procedures are now carried out in service laboratories for other purposes, the identification of micro-organisms has not heretofore enjoyed the benefits of automation.
Inasmuch as the present invention provides a technique for facilitating the identification of micro-organisms by creating for each organism a machine-readable identifier which is indirectly derived from the genetic code implanted therein, we shall now briefly review the fundamental nature of micro-organisms.
The cell is the basic unit of life both in terms of structure and function. A bacterium is a single-celled micro-organism in which the cell not only is the structural unit but the entire organism. In contradistinction, in multicellular organisms, the cells are combined into units which are integrated into a system which constitutes the living organism. A virus, on the other hand, is a parasite; for it is obligated to grow within an appropriate host cell and cannot multiply outside this cell. When a virus enters a living cell, it is capable of creating hundreds of identical particles, the virus exploiting the host cell's energy and biological mechanism for this purpose.
A typical cell is enclosed by a membrane within which is contained a nucleus, the differentiated nucleoprotein-rich protoplasm of the cell. The living matter between the cell membrane and the nucleus is the cytoplasm which incorporates various structural or particulate entities. The term "bacterium" is applicable to any of a group of diverse procaryolic single-celled oganisms; that is, a cell having no internal membranes which separate the nucleus from the cytoplasm.
The nuclear material or DNA (deoxyribonucleic acid), the chemical substance responsible for the transmission of hereditary data, occupies a position near the centre of a bacterial cell. This material is the entire genetic apparatus of the bacterium and consists of a single, circular chromosome to which all genes are linked, each gene being a repository of a unit of genetic information. DNA is a long, ropelike molecule consisting of two strands, each wound about the other to form a double helix.
Also present in the cell is another type of nucleic acid, RNA (ribonucleic acid). RNA, which is composed of a single strand, acts to process the information coded in DNA for protein synthesis. Such synthesis is effected by ribosomes, which are large RNA protein particles in the cytoplasm of the bacterial cell. Before protein synthesis can take place, the code borne by DNA must first be exported to a substance that conveys information from the DNA in the nuclear region to the ribosomes in the cytoplasm. This substance is known as messenger RNA or mRNA.
The process by which a complementary single-stranded mRNA is synthesized from one of the DNA strands is referred to as "transcription". Transcription is the first step in gene expression. The next step is translation, the process by which the genetic information now locked in the mRNA molecule governs protein synthesis.
The genetic information or genotype of a cell is determined by data contained in its chromosome which is divided into genes. A gene consists of hundreds of nucleotide pairs and it specifies the formation of a particular polypeptide; e.g., a chain formed by a plurality of amino acids linked by peptide bonds. Thus if a polypeptide chain is constituted by 200 amino acids, then the gene coding for this polypeptide must contain 600 base pairs, three bases for each amino acid. A bacterial chromosome has the capacity to code for about 3000 different proteins, a protein being composed of an extremely large number of amino acids joined by peptide bonds.
In short, the deciphering of the genetic code carried by the DNA and its application to the production of proteins essential for all growth and activity, entail "transcription" to transfer information from DNA to RNA, and "translation" to convert RNA information into protein.
In microbiology, micro-organisms are classified in taxonomic categories, nomenclature being used to name the units delineated and characterized by classification. Identification involves the use of criteria established for classification and nomenclature in order to identify micro-organisms by comparing the characteristics of an unknown unit with known units. Thus with a newly isolated micro-organism, its identification requires an adequate characterization thereof and then a comparison with published descriptions of other similar micro-organisms.
For the reasons set forth above, because existing microbiological identification procedures do not make use of identifiers in the form of a symbol or other indicia, these procedures are time-consuming, labour-intensive and expensive.
With a view to automating the identification of pathological organisms, U.S. Pat. No. 4,288,543 to Sielaff et al discloses a procedure in which the susceptibility of various strains of bacteria to antimicrobial agents is tested, this being done in conjunction with a determination of the light-scattering index of the bacteria being tested. The numerical growth data obtained by the light scatter comparisons are analysed by computer-assisted techniques to identify the strains of bacteria.
The admitted drawback to this patented procedure is that one must use agents not in common therapeutic use in order to avoid errors resulting from strains which have become immune to various therapeutically utilised antibiotic agents. The Sielaff patent also makes of record other patents and publications dealing with the automated, rapid identification of bacteria by computer analysis of growth inhibition patterns. Though these computer-assisted procedures represent, at least in theory, a significant advance over classic procedures for the identification of microbes, the basic problem of identification is not obviated thereby, for the microbes are not supplied with unique markers or identifiers to facilitate their positive identification.